WO2020077756A1 - Séquence codante de protéine nd4 et application correspondante - Google Patents

Séquence codante de protéine nd4 et application correspondante Download PDF

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WO2020077756A1
WO2020077756A1 PCT/CN2018/118662 CN2018118662W WO2020077756A1 WO 2020077756 A1 WO2020077756 A1 WO 2020077756A1 CN 2018118662 W CN2018118662 W CN 2018118662W WO 2020077756 A1 WO2020077756 A1 WO 2020077756A1
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sequence
nucleic acid
protein
cells
vector
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PCT/CN2018/118662
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English (en)
Chinese (zh)
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李斌
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武汉纽福斯生物科技有限公司
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Priority to KR1020247001775A priority Critical patent/KR20240014102A/ko
Priority to KR1020217001385A priority patent/KR102627561B1/ko
Priority to CN202110786772.2A priority patent/CN113528510A/zh
Priority to CN202110786630.6A priority patent/CN113476484A/zh
Priority to SG11202012044QA priority patent/SG11202012044QA/en
Priority to MX2020013772A priority patent/MX2020013772A/es
Priority to CA3103740A priority patent/CA3103740A1/fr
Priority to JP2021521870A priority patent/JP2021529001A/ja
Priority to AU2019296451A priority patent/AU2019296451B2/en
Priority to CN201980003485.0A priority patent/CN110876269B/zh
Priority to PCT/CN2019/094136 priority patent/WO2020001657A1/fr
Priority to BR112020026361-3A priority patent/BR112020026361A2/pt
Priority to EP19826653.8A priority patent/EP3814492A4/fr
Priority to CA3109432A priority patent/CA3109432A1/fr
Priority to EP19853225.1A priority patent/EP3840785A4/fr
Priority to JP2021509893A priority patent/JP7403852B2/ja
Priority to CN201980054770.5A priority patent/CN112584874A/zh
Priority to PCT/CN2019/101538 priority patent/WO2020038352A1/fr
Priority to KR1020217007727A priority patent/KR20210068014A/ko
Priority to SG11202101032VA priority patent/SG11202101032VA/en
Priority to AU2019323434A priority patent/AU2019323434A1/en
Priority to US16/836,644 priority patent/US11034954B2/en
Publication of WO2020077756A1 publication Critical patent/WO2020077756A1/fr
Priority to US17/181,849 priority patent/US11352645B2/en
Priority to US17/317,295 priority patent/US20220340895A1/en
Priority to US17/320,388 priority patent/US11332741B1/en
Priority to AU2021204690A priority patent/AU2021204690A1/en
Priority to US17/726,833 priority patent/US20220259619A1/en
Priority to JP2023029170A priority patent/JP2023078173A/ja
Priority to JP2023205807A priority patent/JP2024028861A/ja
Priority to AU2023285773A priority patent/AU2023285773A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y106/00—Oxidoreductases acting on NADH or NADPH (1.6)
    • C12Y106/99—Oxidoreductases acting on NADH or NADPH (1.6) with other acceptors (1.6.99)
    • C12Y106/99003—NADH dehydrogenase (1.6.99.3)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to the field of biological preparations, in particular to the coding sequence of ND4 protein and its application.
  • Leber hereditary optic neuropathy is a degenerative vision disorder, usually manifested as bilateral loss of central vision. The average age of onset is in the middle of 20 years, usually no pain within a few weeks to several months, until the vision of both eyes deteriorates below 0.1, which seriously affects the patient's quality of life.
  • LHON is caused by a mutation in the mitochondrial gene and is related to a mutation in one of the three mitochondrial genes of NADH ubiquinone oxidoreductase, the complex I subunit of the mitochondrial respiratory chain.
  • G3460A mutation that affects the ND1 gene the T14484C mutation that affects the ND6 gene, and the G11778A mutation that affects the ND4 gene are considered to be the main causes of LHON, and each mutation has a significant risk of permanent vision loss. All of these are related to the focal degeneration of retinal ganglion cells.
  • CN 102634527 B discloses a recombinant human NADH dehydrogenase subunit 4 protein gene (ND4 gene) and its expression vector construction method.
  • COX10 encodes a 28 amino acid peptide chain to guide the ND4 protein into mitochondria in.
  • CN104450747A discloses a recombinant adeno-associated virus-NADH dehydrogenase subunit 4 (ND4) full-length gene and a medicament for treating Leber hereditary optic neuropathy.
  • the gene consists of CAG promoter sequence, ND4 coding sequence with COX10 mitochondrial localization sequence and UTR.
  • the purpose of the present invention is to provide an expression system and preparation method of human NADH dehydrogenase subunit 4 protein with high transfection efficiency and good therapeutic effect.
  • the object of the present invention is to provide an optimized nucleic acid sequence, vector and preparation method encoding human NADH dehydrogenase subunit 4 protein.
  • nucleotide sequence encoding the human NADH dehydrogenase subunit 4 protein, and the nucleotide sequence is selected from the group consisting of:
  • nucleotide sequence has ⁇ 95% identity with the nucleotide sequence shown in SEQ ID NO.:1, preferably ⁇ 98%, more preferably ⁇ 99%;
  • the nucleotide sequence includes a DNA sequence, a cDNA sequence, or an mRNA sequence.
  • the nucleotide sequence includes a single-stranded sequence and a double-stranded sequence.
  • the nucleotide sequence includes a nucleotide sequence that is completely complementary to SEQ ID NO.:1.
  • a fusion nucleic acid comprising the nucleotide sequence encoding the human NADH dehydrogenase subunit 4 protein according to the first aspect of the present invention.
  • the fusion nucleic acid further comprises a sequence selected from the group consisting of a mitochondrial targeting peptide coding sequence, a UTR sequence, or a combination thereof.
  • the coding sequence of the mitochondrial targeting peptide includes: COX10 sequence and / or OPA1 sequence.
  • the COX10 coding sequence has the sequence shown in SEQ ID NO .: 2.
  • the coding sequence of OPA1 has the sequence shown in SEQ ID NO.:3.
  • the UTR sequence includes 3'-UTR and / or 5'-UTR, preferably 3'-UTR.
  • the UTR sequence has the sequence shown in SEQ ID NO .: 4 or 11.
  • the fusion nucleic acid has a structure of formula I from the 5 'end to the 3' end:
  • Each "-" is independently a bond or nucleotide linking sequence
  • Z0 is none, or 5'-UTR sequence
  • Z1 is the coding sequence of mitochondrial targeting peptide
  • Z2 is the nucleotide sequence according to the first aspect of the present invention.
  • Z3 is a 3'-UTR sequence.
  • the Z1 is a COX10 coding sequence or an OPA1 coding sequence.
  • the structure of the fusion nucleic acid from the 5'-3 'end is COX10-ND4-UTR.
  • sequence of the fusion nucleic acid is shown in SEQ ID NO .: 5; wherein,
  • Bits 1-84 are the COX10 coding sequence
  • Position 85-1464 is the nucleotide sequence encoding human NADH dehydrogenase subunit 4 protein
  • Positions 1465-2889 are 3'-UTR sequences.
  • the structure of the fusion nucleic acid from the 5'-3 'end is OPA1-ND4-UTR.
  • the fusion nucleic acid sequence is shown in SEQ ID NO.:10.
  • sequence is as shown in SEQ ID NO .: 10 in the fusion nucleic acid
  • Bit 1-266 is the coding sequence of OPA1;
  • Positions 267-1646 are the nucleotide sequence encoding human NADH dehydrogenase subunit 4 protein
  • Positions 1647-2271 are 3'-UTR sequences.
  • each nucleotide linking sequence is 1-30 nt, preferably 1-15 nt, more preferably 3-6 nt.
  • the nucleotide linking sequence is derived from a nucleotide linker sequence formed by restriction enzyme digestion.
  • a vector containing the nucleotide sequence according to the first aspect of the present invention or the fusion nucleic acid according to the second aspect of the present invention is provided.
  • the vector comprises one or more promoters, the promoter is operable with the nucleic acid sequence, enhancer, transcription termination signal, polyadenylation sequence, origin of replication, selectable marker , Nucleic acid restriction sites, and / or homologous recombination sites.
  • the vector is selected from the group consisting of plasmids and viral vectors.
  • the vector is selected from the group consisting of a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, or a combination thereof.
  • the vector is an AAV vector.
  • the serotype of the AAV vector is selected from: AAV2, AAV5, AAV7, AAV8, or a combination thereof.
  • the vector includes DNA virus and retrovirus vector.
  • the vector is an AAV vector containing or inserted with the nucleotide sequence as described in the first aspect of the invention or the fusion nucleic acid as described in the second aspect of the invention; preferably, the AAV vector plasmid pSNaV .
  • the backbone of the vector is adeno-associated virus vector plasmid pSNaV.
  • the vector is used to express recombinant human NADH dehydrogenase subunit 4 protein.
  • a host cell containing the vector according to the third aspect of the present invention, or a foreign nucleotide as described in the first aspect of the present invention integrated into its chromosome Sequence or fusion nucleic acid according to the second aspect of the invention.
  • the host cell is a mammalian cell, and the mammal includes human and non-human mammals.
  • the host cell is selected from the group consisting of HEK293 cells, photoreceptor cells (including cone cells and / or rod cells), other visual cells (such as biganglionic cells), (optical) nerve cells, Or a combination thereof.
  • the host cell is selected from the group consisting of rod cells, cone cells, light-giving bipolar cells, light-extracting bipolar cells, horizontal cells, ganglion cells, amacrine cells, or combination.
  • the host cell is a (retinal) ganglion cell.
  • a fifth aspect of the present invention there is provided the use of the carrier according to the third aspect of the present invention, for preparing a formulation or composition for restoring the vision of a subject and / or treating the eye disease.
  • the eye disease is a degenerative disease of the optic nerve.
  • the preparation or composition is used to treat focal degeneration of retinal ganglion cells.
  • the formulation or composition is used to treat hereditary optic neuropathy, preferably Leber's hereditary optic neuropathy (LHON).
  • hereditary optic neuropathy preferably Leber's hereditary optic neuropathy (LHON).
  • a pharmaceutical preparation comprising (a) the carrier according to the third aspect of the present invention, and (b) a pharmaceutically acceptable carrier or excipient.
  • the dosage form of the pharmaceutical preparation is selected from the group consisting of a lyophilized preparation, a liquid preparation, or a combination thereof.
  • the vector is selected from the group consisting of a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, or a combination thereof.
  • the vector is an AAV vector.
  • the content of the carrier in the pharmaceutical preparation is 1 ⁇ 10 9 -1 ⁇ 10 16 , preferably 1 ⁇ 10 12 -1 ⁇ 10 13 viruses / ml.
  • the pharmaceutical preparation is used to treat ocular diseases, preferably to treat degenerative diseases of the optic nerve, and more preferably to treat focal degeneration of retinal ganglion cells.
  • the pharmaceutical preparation is used to treat hereditary optic neuropathy, preferably Leber's hereditary optic neuropathy (LHON).
  • hereditary optic neuropathy preferably Leber's hereditary optic neuropathy (LHON).
  • a method of treatment comprising applying the vector of the third aspect of the present invention to a subject in need.
  • the vector is selected from the group consisting of a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, or a combination thereof.
  • the vector is an AAV vector.
  • the carrier is introduced into the eyes of the desired subject.
  • the objects in need include human and non-human mammals.
  • the treatment method is a method for treating eye diseases.
  • the eye disease is hereditary optic neuropathy, preferably Leber's hereditary optic neuropathy (LHON).
  • LHON Leber's hereditary optic neuropathy
  • An eighth aspect of the present invention provides a method for preparing recombinant human NADH dehydrogenase subunit 4 protein, comprising the steps of: cultivating the host cell of the fourth aspect of the present invention to obtain a recombinant human NADH dehydrogenase subunit 4 protein.
  • the ninth aspect of the present invention provides a fusion protein encoded by the fusion nucleic acid according to the second aspect of the present invention.
  • Figure 1 shows the nucleotide sequence comparison of the ND4 fusion nucleic acid and the optimized ND4 fusion nucleic acid.
  • the "ND4 gene” line sequence is an unoptimized ND4 fusion nucleic acid sequence
  • the "human optimized ND4 gene” line sequence is an optimized ND4 fusion nucleic acid sequence.
  • Figure 2 shows the results of PCR nucleic acid electrophoresis to verify ND4 (lane A) and optimized ND4 (lane B) gene cloning results.
  • Figure 3 shows the fluorescence observation results of rAAV2-ND4 (A) and rAAV2-optimized ND4 (B) infected 293T cells.
  • Fig. 4 shows the expression levels of rAAV2-optimized ND4 and rAAV2-ND4 on 293T cells using ⁇ -actin as the internal reference protein.
  • Lane A is rAAV2-ND4 and lane B is rAAV2-optimized ND4.
  • Figure 5 shows the expression level of ND4 protein after ⁇ -actin as the internal reference protein, rAAV2-optimized ND4 and rAAV2-ND4 respectively infected 293T cells.
  • Fig. 6 shows the expression levels of rAAV2-optimized ND4 and rAAV2-ND4 on rabbit optic nerve cells using ⁇ -actin as the internal reference protein.
  • the lane A is rAAV2-optimized ND4 and lane B is rAAV2-ND4.
  • Figure 7 shows the expression level of ND4 protein after ⁇ -actin as the internal reference protein, rAAV2-optimized ND4 and rAAV2-ND4 respectively infect the rabbit optic nerve cells.
  • Figure 8 shows a fundus photograph taken under a rabbit eye glass microscope, where A is the injection of rAAV2-ND4 virus and B is the injection of rAAV2-optimized ND4 virus.
  • Fig. 9 shows the results of microscopic examination of HE slices in rabbit eyes, where A is the injection of rAAV2-ND4 virus and B is the injection of rAAV2-optimized ND4 virus.
  • the present inventors targeted and optimized the coding sequence of the recombinant human NADH dehydrogenase subunit 4 protein gene coding, thereby obtaining a highly efficient transcription in mammalian (such as human) cells And highly express the nucleotide sequence and fusion nucleic acid of ND4 protein, and construct a recombinant expression vector of recombinant human NADH dehydrogenase subunit 4 protein.
  • the transcription efficiency and translation efficiency of the ND4 coding sequence SEQ ID NO.:1 were significantly improved, and the expression of recombinant human NADH dehydrogenase subunit 4 protein was increased by more than 3 times.
  • the term “about” may refer to a value or composition within an acceptable error range for a particular value or composition determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.
  • the expression “about 100” includes all values between 99 and 101 and (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the terms "containing” or “including (including)” may be open, semi-closed, and closed. In other words, the term also includes “consisting essentially of” or “consisting of”.
  • Sequence identity by comparing two aligned along a predetermined comparison window (which can be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the reference nucleotide sequence or protein) Sequence and determine the number of positions where the same residue appears. Generally, this is expressed as a percentage.
  • a predetermined comparison window which can be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the reference nucleotide sequence or protein
  • the terms “subject” and “subject in need” refer to any mammal or non-mammal. Mammals include but are not limited to humans, vertebrates such as rodents, non-human primates, cattle, horses, dogs, cats, pigs, sheep, goats.
  • Adeno-associated virus also known as adeno-associated virus, belongs to the genus of dependent viruses of the family Parvoviridae. It is the simplest single-stranded DNA-defective virus found so far and requires a helper virus (usually an adeno-associated virus). Virus) involved in replication. It encodes the cap and rep genes in the inverted repeat sequence (ITR) at both ends. ITRs play a decisive role in virus replication and packaging. The cap gene encodes a viral capsid protein, and the rep gene is involved in virus replication and integration. AAV can infect a variety of cells.
  • Recombinant adeno-associated virus vector is derived from non-pathogenic wild-type adeno-associated virus. Due to its good safety, wide host cell range (dividing and non-dividing cells), low immunogenicity, the time to express foreign genes in vivo The long-term characteristics are regarded as one of the most promising gene transfer vectors, and are widely used in gene therapy and vaccine research worldwide. After more than 10 years of research, the biological characteristics of recombinant adeno-associated virus have been thoroughly understood, especially its application in various cells, tissues and in vivo experiments has accumulated a lot of data.
  • rAAV is used in gene therapy research of various diseases (including in vivo and in vitro experiments); at the same time, as a characteristic gene transfer vector, it is also widely used in gene function research, disease model construction, gene preparation Knock out rats and so on.
  • the vector is a recombinant AAV vector.
  • AAVs are relatively small DNA viruses that can integrate into the genome of the cells they infect in a stable and site-specific manner. They can infect a large series of cells without any effect on cell growth, morphology or differentiation, and they do not seem to involve human pathology.
  • the AAV genome has been cloned, sequenced and characterized.
  • the AAV contains about 4700 bases and contains about 145 base inverted terminal repeat (ITR) regions at each end, which serve as the origin of replication of the virus.
  • ITR inverted terminal repeat
  • the rest of the genome is divided into two important regions with capsidization functions: the left part of the genome containing the rep gene involved in viral replication and viral gene expression; and the right part of the genome containing the cap gene encoding the viral capsid protein.
  • AAV vectors can be prepared using standard methods in the art. Adeno-associated viruses of any serotype are suitable. Methods for purifying vectors can be found in, for example, U.S. Patent Nos. 6,566,118, 6,989,264, and 6,995,006, the disclosures of which are incorporated by reference in their entirety. The preparation of hybrid carriers is described in, for example, PCT Application No. PCT / US2005 / 027091, the disclosure content of which is incorporated herein by reference in its entirety. The use of vectors derived from AAV for in vitro and in vivo gene transfer has been described (see, eg, International Patent Application Publication Nos. WO91 / 18088 and WO93 / 09239; US Patent Nos.
  • Replication-defective recombinant AAV can be prepared by co-transfecting the following plasmids into a cell line infected with a human helper virus (eg, adenovirus): the nucleic acid sequence of interest is flanked by two AAV inverted terminal repeat sequences (ITR) Plasmids in the region, and plasmids carrying AAV capsidization genes (rep and cap genes). The resulting AAV recombinants are then purified by standard techniques.
  • a human helper virus eg, adenovirus
  • the recombinant vector is capsidized into viral particles (for example, including but not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15 And AAV16 AAV virions). Therefore, the present disclosure includes recombinant viral particles (recombinant because they contain recombinant polynucleotides) containing any of the vectors described herein. Methods for generating such particles are known in the art and are described in US Patent No. 6,596,535.
  • the technical problem to be solved by the present invention is to overcome the technical defects in the prior art that NADH dehydrogenase subunit 4 protein transfection efficiency is not high and the therapeutic effect is not good.
  • the invention provides a targeted NADH dehydrogenase subunit 4 protein, and a preparation method and application thereof. It has been found through research that the optimized ND4 gene sequence (SEQ ID NO .: 1) of the present invention makes ND4 protein expression more efficient, and more ND4 protein plays a physiological role in the optic ganglion cells of patients.
  • the nucleotide sequence of the nucleic acid encoding human NADH dehydrogenase subunit 4 protein according to the present invention is shown in SEQ ID NO .: 1.
  • the nucleic acid encoding human NADH dehydrogenase subunit 4 protein has a total length of 1380 bp.
  • the nucleic acid encoding human NADH dehydrogenase subunit 4 protein is also called ND4 optimized gene or ND4 optimized nucleic acid.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • the nucleotide is DNA.
  • DNA forms include cDNA, genomic DNA, or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • DNA can be a coding strand or a non-coding strand.
  • the full-length nucleotide sequence of the present invention or a fragment thereof can usually be obtained by PCR amplification method, recombination method or artificial synthesis method.
  • primers can be designed according to the related nucleotide sequences disclosed, especially the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared according to a conventional method known to those skilled in the art can be used as Template, the relevant sequence is amplified.
  • the DNA sequence encoding the polypeptide (or fragment or derivative thereof) of the present invention can be obtained completely by chemical synthesis.
  • the DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector or polypeptide coding sequence of the present invention.
  • the above polynucleotide, vector or host cell may be isolated.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in a natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances present in the natural state.
  • nucleotide sequence is shown in SEQ ID NO .: 1.
  • the relevant sequence can be obtained in large quantities by the recombination method. This is usually done by cloning it into a vector, then transferring it into a cell, and then isolating the relevant sequence from the propagated host cell by conventional methods.
  • synthetic methods can also be used to synthesize the relevant sequences, especially when the length of the fragments is short.
  • a long sequence can be obtained by synthesizing multiple small fragments and then connecting them.
  • the method of amplifying DNA / RNA using PCR technology is preferably used to obtain the gene of the present invention.
  • the primers used for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods.
  • the amplified DNA / RNA fragments can be separated and purified by conventional methods such as by gel electrophoresis.
  • the present invention also relates to a vector comprising the polynucleotide of the present invention, and a host cell produced by genetic engineering using the vector or protein coding sequence of the present invention, and a method of using the host cell to express ND4 protein by recombinant technology.
  • the polynucleotide sequences of the present invention can be used to obtain host cells (such as mammalian cells) expressing the ND4 protein of the present invention. Generally, it includes the step of introducing the polynucleotide according to the first aspect of the present invention or the vector according to the third aspect of the present invention into a host cell.
  • an expression vector containing the DNA sequence encoding the polypeptide of the present invention and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombinant technology and so on.
  • the DNA sequence can be effectively linked to an appropriate promoter in an expression vector to guide mRNA synthesis.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green color for eukaryotic cell culture Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green color for eukaryotic cell culture Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • Vectors containing the appropriate DNA sequence and the appropriate promoter or control sequence described above can be used to transform a suitable host cell so that it can express the polypeptide.
  • the host cell may be a prokaryotic cell, or a lower eukaryotic cell, or a higher eukaryotic cell, such as a mammalian cell (including human and non-human mammals). Representative examples are: animal cells such as CHO, NS0, COS7, or 293 cells.
  • animal cells such as CHO, NS0, COS7, or 293 cells.
  • the host cell is selected from the group consisting of rod cells, cone cells, light-giving bipolar cells, light-extracting bipolar cells, horizontal cells, ganglion cells, amacrine cells, or combination.
  • Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • the host is a prokaryotic organism such as E. coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl 2 method.
  • the procedures used are well known in the art.
  • Another method is to use MgCl 2 .
  • transformation can also be carried out by electroporation.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • the obtained transformant can be cultured by a conventional method and express the protein encoded by the gene of the present invention.
  • the medium used in the culture can be selected from various conventional mediums.
  • the cultivation is carried out under conditions suitable for the growth of host cells.
  • the selected promoter is induced by an appropriate method (such as temperature conversion or chemical induction), and the cell is cultured for a period of time.
  • the polypeptide in the above method may be expressed in a cell, on a cell membrane, or secreted out of the cell. If necessary, the protein can be separated and purified by various separation methods using its physical, chemical and other characteristics. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with protein precipitation agent (salting out method), centrifugation, osmotic disruption, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment treatment with protein precipitation agent (salting out method)
  • centrifugation osmotic disruption
  • ultra-treatment ultra-centrifugation
  • molecular sieve chromatography gel filtration
  • adsorption layer Analysis ion exchange chromatography
  • HPLC high performance
  • the present invention changes the COX10 mitochondrial coding sequence to a nucleic acid coding sequence (as shown in SEQ ID NO: 2) and performs ND4 nucleotide sequence optimization (as shown in SEQ ID NO: 1, in the present invention is referred to as optimizing ND4 gene / Nucleic acid), this sequence is specially optimized, and the transcription efficiency and translation efficiency are significantly improved.
  • the homology of the optimized COX10 + ND4 and unoptimized COX10 + ND4 sequences is 76.16% (1115/1464).
  • an optimized coding sequence of recombinant human NADH dehydrogenase subunit 4 protein with high transcription efficiency and translation efficiency is provided.
  • the coding sequence is shown in SEQ ID NO .: 1.
  • optically ND4 coding sequence and “optimized ND4 coding gene” both refer to a nucleotide sequence used to encode recombinant human NADH dehydrogenase subunit 4 protein, and the nucleotide sequence Encoding the amino acid sequence shown in SEQ ID NO.:1.
  • the present invention also provides a fusion nucleic acid comprising the nucleic acid encoding the human NADH dehydrogenase subunit 4 protein.
  • fusion nucleic acid refers to a nucleic acid composed of two or more nucleotide sequences from different sources, or two or more nucleosides from the same source but whose natural positions are not connected to each other Nucleic acid formed by linking acid sequences.
  • the protein encoded by the fusion nucleic acid of the present invention is called a fusion protein. In the present invention, it is an ND4 optimized protein.
  • the fusion nucleic acid is operably linked to the coding sequence and / or UTR sequence of the mitochondrial targeting peptide in the nucleic acid encoding the human NADH dehydrogenase subunit 4 protein.
  • the coding sequence of the mitochondrial targeting peptide is as shown in SEQ ID NO .: 2
  • the coding sequence of the COX10, the nucleic acid encoding the human NADH dehydrogenase subunit 4 protein, and the UTR sequence are sequentially arranged from the 5 ′ end to the 3 ′ end .
  • the sequence of the fusion nucleic acid is shown in SEQ ID NO .: 5.
  • the nucleotide sequence of the fusion nucleic acid has a full length of 2889 bp, from 1 bp to 84 bp is the optimized COX10 sequence (a total of 84 bp); 85 bp to 1464 bp is the optimized ND4 gene, that is, the encoding human NADH
  • the nucleic acid of the hydrogenase subunit 4 protein (total 1380bp), the position from 1465bp to 2889bp is the UTR sequence (total 1425bp, also known as 3'UTR).
  • the COX10 sequence guides the ND4 protein into the mitochondria and exerts its physiological functions; 3'UTR is a non-coding sequence designed behind the ND4 protein, and its role is to stabilize the coding sequence of the mitochondrial targeting peptide and the expression of ND4. Among them, the homology of the optimized COX10 + ND4 and unoptimized COX10 + ND4 sequences is 76.16%.
  • sequence of the fusion nucleic acid is shown in SEQ ID NO.:10.
  • the invention also provides an expression vector for ND4 protein, which contains the optimized ND4 coding sequence of the invention.
  • sequence information By providing the sequence information, skilled artisans can use available cloning techniques to generate nucleic acid sequences or vectors suitable for transduction into cells.
  • the nucleic acid sequence encoding the ND4 protein is provided as a vector, preferably an expression vector.
  • it can be provided as a gene therapy vector that is preferably suitable for transduction and expression in retinal target cells.
  • the vector may be viral or non-viral (eg plasmid).
  • Viral vectors include those derived from the following: adenovirus, adeno-associated virus (AAV), retrovirus, lentivirus, herpes virus, vaccinia virus, MMLV, GaLV, simian immunodeficiency virus (SIV) including mutant forms , HIV, pox virus and SV40.
  • the viral vector is replication-defective, although it is envisaged that it may be replication-deficient, capable of replication, or conditionally replicated.
  • Viral vectors can usually maintain an extrachromosomal state without integrating into the genome of the target retinal cell.
  • a preferred viral vector for introducing ND4 protein-encoding nucleic acid sequences into retinal target cells is an AAV vector, such as a self-complementary adeno-associated virus (scAAV).
  • Selective targeting can be achieved using specific AAV serotypes (AAV serotype 2 to AAV serotype 12) or modified versions of any of these serotypes (including AAV 4YF and AAV 7m8 vectors).
  • the viral vector can be modified to delete any non-essential sequences.
  • viruses can be modified to delete all or part of the IX gene, Ela and / or Elb genes.
  • helper viruses such as adenovirus
  • replication is very inefficient.
  • the replication gene and capsid gene are provided in trans (in the pRep / Cap plasmid), and only the 2ITR of the AAV genome is retained and packaged into the virion, while the adenovirus gene Provided by adenovirus or another plasmid. Similar modifications can also be made to lentiviral vectors.
  • Viral vectors have the ability to enter cells.
  • non-viral vectors such as plasmids can be complexed with agents to facilitate the uptake of viral vectors by target cells.
  • agents include polycationic agents.
  • delivery systems such as liposome-based delivery systems can be used.
  • the carrier for use in the present invention is preferably suitable for use in vivo or in vitro, and preferably suitable for use in humans.
  • the vector will preferably contain one or more regulatory sequences to direct the expression of the nucleic acid sequence in retinal target cells. Regulatory sequences can include promoters, enhancers, transcription termination signals, polyadenylation sequences, origins of replication, nucleic acid restriction sites, and homologous recombination sites operably linked to the nucleic acid sequence.
  • the vector may also include a selectable marker, for example, to determine the expression of the vector in a growth system (eg, bacterial cells) or in retinal target cells.
  • “Operably linked” means that nucleic acid sequences are functionally related to their operably linked sequences so that they are linked in such a way that they affect each other's expression or function.
  • a nucleic acid sequence operably linked to a promoter will have an expression pattern affected by the promoter.
  • the promoter mediates the expression of the nucleic acid sequence linked thereto.
  • the promoter may be constitutive or may be inducible. Promoters can direct ubiquitous expression in inner retinal cells, or neuron-specific expression. In the latter case, the promoter can direct cell type-specific expression, for example, to the optic ganglion cells. Suitable promoters will be known to those skilled in the art.
  • a suitable promoter may be selected from the group consisting of: L7, thy-1, restorer protein, calcium binding protein, human CMV, GAD-67, chicken ⁇ -actin, hSyn, Grm6, Grm6 enhancer SV40 fusion protein .
  • Targeting can be achieved using cell-specific promoters, such as Grm6-SV40 for selective targeting to optic nerve cells.
  • the Grm6 promoter is a fusion of the 200 base pair enhancer sequence of the Grm6 gene and the SV40 eukaryotic promoter.
  • the Grm6 gene encodes a specific metabolic glutamate receptor mGluR6 for optic nerve cells.
  • the preferred sources of the Grm6 gene are mice and humans.
  • Ubiquitous neuronal promoters can be used to achieve ubiquitous expression, examples of which are known and available in the art.
  • One such example is CAG.
  • the CAG promoter is then a fusion of the early CMV enhancer and the chicken ⁇ -actin promoter.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • the promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto.
  • Another example of a suitable promoter is elongation growth factor-1 ⁇ (EF-1 ⁇ ).
  • constitutive promoter sequences can also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse breast cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Russ sarcoma virus promoter, and human gene promoters, such as but not limited to the actin promoter , Myosin promoter, heme promoter and creatine kinase promoter.
  • the present invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered as part of the present invention.
  • an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or turn off the expression when expression is undesirable.
  • inducible promoters include, but are not limited to, metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.
  • ND4 protein can be used to express in mammalian cells (preferably human, more preferably human optic nerve cells or photoreceptor cells).
  • mammalian cells preferably human, more preferably human optic nerve cells or photoreceptor cells.
  • the present invention preferably uses an adeno-associated virus as an expression vector.
  • the present invention also provides a host cell for expressing ND4 protein.
  • the host cell is a mammalian cell (preferably a human, more preferably a human optic nerve cell or a photoreceptor cell) to increase the expression level of ND4 protein.
  • the present invention provides a formulation or composition comprising (a) the carrier according to the third aspect of the present invention, and (b) a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical preparation is used to treat eye diseases.
  • the pharmaceutical preparation is used to treat hereditary optic neuropathy, preferably Leber's hereditary optic neuropathy (LHON).
  • hereditary optic neuropathy preferably Leber's hereditary optic neuropathy (LHON).
  • the “active ingredient” in the pharmaceutical composition of the present invention refers to the vector of the present invention, such as a viral vector (including adeno-associated viral vector).
  • the "active ingredients", formulations and / or compositions described in the present invention can be used to treat eye diseases.
  • Safe and effective amount means that the amount of the active ingredient is sufficient to significantly improve the condition or symptoms without causing serious side effects.
  • “Pharmaceutically acceptable carrier or excipient” means: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficient Low toxicity.
  • “Compatibility” here means that the components of the composition can be blended with the active ingredient of the present invention and between them without significantly reducing the efficacy of the active ingredient.
  • the composition may be liquid or solid, such as a powder, gel or paste.
  • the composition is a liquid, preferably an injectable liquid. Suitable excipients will be known to those skilled in the art.
  • the carrier can be administered to the eye by subretinal or intravitreal administration.
  • the carrier is provided as an injectable liquid.
  • the injectable liquid is provided as a capsule or syringe.
  • Examples of pharmaceutically acceptable carrier parts are cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid) , Magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as ), Wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
  • cellulose and its derivatives such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.
  • gelatin such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate,
  • the composition may contain physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.
  • the nucleic acid or fusion nucleic acid encoding ND4 provided by the present invention can produce ND4 protein or ND4 fusion protein in vitro or in vivo, and the fusion protein or the preparation containing the fusion protein can be applied to the preparation of a medicament for treating Leber hereditary optic neuropathy.
  • the optimized nucleic acid encoding human NADH dehydrogenase subunit 4 protein has a higher expression level, resulting in more ND4 fusion proteins being translated, and the COX10 sequence can accurately locate the ND4 fusion protein on the mitochondrial inner membrane, so there are more Many ND4 proteins were transfected into mitochondria.
  • An agent containing COX10-optimized ND4 fusion nucleic acid was injected into the vitreous cavity of the rabbit eye. The agent remained viable in the vitreous cavity and was transfected into optic nerve cells.
  • the optimized ND4 nucleic acid encodes more ND4 protein than the prior art, its transfection efficiency is higher, and it can better treat Leber's hereditary optic neuropathy.
  • the present invention mainly has the following advantages:
  • the gene sequence encoding the recombinant human NADH dehydrogenase subunit 4 protein (ND4) of the present invention has been specially optimized. Compared with the unoptimized DNA coding sequence of ND4, the transcription efficiency and translation efficiency are significantly improved. The optimized sequence ND4 protein expression was significantly increased and the biological activity was high.
  • the optimized COX10 sequence or OPA1 sequence of the present invention can accurately locate the ND4 fusion protein on the mitochondrial inner membrane, so more ND4 protein is transfected into the mitochondria.
  • the optimized ND4 encoding gene (SEQ ID NO .: 1) or fusion nucleic acid of the present invention can very effectively treat Leber's hereditary optic neuropathy and has good safety.
  • ND4 nucleotide sequence (National Biotechnology Information Center reference sequence: yp_003024035.1)
  • the present invention changes the COX10 mitochondrial coding sequence to a nucleic acid coding sequence (as shown in SEQ ID NO: 2 )
  • ND4 nucleotide sequence optimization (as shown in SEQ ID NO: 1, referred to as optimizing ND4 gene / nucleic acid in the present invention), this sequence is specially optimized, the transcription efficiency and translation efficiency are significantly improved, and the optimized COX10 +
  • the homology between ND4 and the unoptimized COX10 + ND4 sequence is 76.16%.
  • the UTR sequence (shown as SEQ ID NO: 4) is connected to the 3 'end of the optimized ND4 gene, and the sequence of the fusion gene (or fusion nucleic acid) is shown as SEQ ID NO .: 5, which is provided by Chengdu Qingke Zixi Synthesized by Biotechnology Co., Ltd.
  • pAAV2-ND4 For the preparation of the control pSNaV / rAAV2 / 2-ND4 (hereinafter referred to as pAAV2-ND4), see CN 102634527 B.
  • HEK293 cells were seeded in a 225cm 2 cell culture flask with a density of 3.0 ⁇ 10 7 cells / mL, the medium was DMEM + 10% bovine serum, and 37% 5% CO 2 Incubate in the incubator overnight. The medium was changed on the day of transfection, and culture was continued with fresh DMEM medium containing 10% bovine serum. When the cells grow to 80-90%, the culture medium is discarded and pAAV2-ND4 and pAAV2-optimized ND4 are transfected with the PlasmidTrans II (VGTC) transfection kit (for specific transfection steps, see CN 102634527 B Example 1). 48 hours after transfection, cells were collected.
  • VGTC PlasmidTrans II
  • Virus collection 1) Prepare dry ice ethanol bath (or liquid nitrogen) and 37 ° C water bath; 2) Collect the toxin-producing cells together with the culture medium into a 15ml centrifuge tube; 3) 1000rpm / min, centrifuge Separate the cells and supernatant for 3 minutes, store the supernatant separately, and resuspend the cells in 1ml of PBS; 4) Repeat the transfer of the cell suspension in a dry ice ethanol bath and a 37 ° C water bath. Minutes, a little shock after each melt.
  • Virus purification 1) Add solid CsCl to the virus concentrate until the density is 1.41g / ml (refractive index is 1.372); 2) Add the sample to the ultracentrifuge tube and use the pre-made 1.41g / The mlCsCl solution fills the remaining space of the centrifuge tube; 3) Centrifuge at 175,000g for 24 hours to form a density gradient. Collect samples of different densities in order and take samples for titer determination. Collect the fraction enriched with rAAV2 particles; 4) Repeat the above process once. The virus was loaded into a 100kDa dialysis bag and dialyzed against desalting at 4 ° C overnight.
  • OPA1 (sequence shown in SEQ ID NO .: 3) can also be fused with the optimized ND4 gene of the present invention, and the coding sequence of the mitochondrial targeting peptide OPA1 can optimize ND4 gene expression
  • the protein is brought into the inner mitochondrial membrane, so as to achieve targeted expression of the protein mitochondria.
  • rAAV2-ND4-EGFP The virus solutions rAAV2-ND4-EGFP and rAAV2-optimized ND4-EGFP were prepared, and the experimental method was the same as in Example 1. Among them, rAAV2-ND4-EGFP is rAAV2-ND4 virus with EGFP tag, rAAV2-optimized ND4-EGFP is rAAV2-optimized ND4 virus with EGFP tag.
  • the frozen 293T cells After recovering the frozen 293T cells, they were subcultured and grown to about 90% of the T75 flask. The cells were digested with trypsin, the cell pellet was taken, and the cell density was resuspended in DMEM complete medium at 5 ⁇ 10 4 cells / mL. Use 96-well plates to plant cells, add 100 ⁇ L of cell suspension to each well, about 5000 cells / well.
  • rAAV2-ND4 and rAAV2-optimized ND4 (1 ⁇ 10 10 vg / 0.05mL) and PBS were punctured the flat part of the ciliary body at 3 mm from the limbus to enter the vitreous cavity. After intravitreal injection, slit lamp, fundus photography and HE staining were performed. After 30 days of injection, RT-PCR and immunoblotting were performed in each group.
  • RNA and reverse transcription of 293T cells transfected with rAAV2-ND4 and rAAV2-Optimized-ND4 and rabbit optic nerve cells were extracted, and the total RNA was extracted using TRIZOL kit and synthesized by reverse transcription.
  • ⁇ -actin-S CGAGATCGTGCGGGACAT (the sequence is shown in SEQ ID NO: 6);
  • ⁇ -actin-A CAGGAAGGAGGGCTGGAAC (the sequence is shown in SEQ ID NO: 7);
  • ND4-S GCCAACAGCAACTACGAGC (sequence shown in SEQ ID NO: 8);
  • ND4-A TGATGTTGCTCCAGCTGAAG (the sequence is shown in SEQ ID NO: 9);
  • Real-time PCR detection was performed on the Real-time PCR Detection System instrument. Add 12.5 ⁇ L of SYBR Green mix, 8 ⁇ L of ddH 2 O, 1 ⁇ L of each primer to a 0.2 mL PCR reaction tube, 2.5 ⁇ L of cDNA sample, and 25 ⁇ L of the total system. Each sample is used to amplify both the target gene and the internal reference gene ⁇ -actin, and the amplification of each gene is repeated three times. In order to reduce the error during the actual sample loading, the reagents shared in each PCR reaction tube can be added together and then packed separately. After the sample is added, fluorescence quantitative PCR is performed.
  • the relative quantitative method is used to study the difference in gene expression. This method does not need to make a standard curve. With ⁇ -actin as the internal reference gene, the analysis software that comes with the instrument can automatically generate expression values and calculate relative expression.
  • the ND4 protein of 293T cells transfected with rAAV2-ND4 and rAAV2-optimized-ND4 and rabbit nerve cells were extracted, and then subjected to 10% polyacrylamide gel electrophoresis, and the spots were transferred to a polyfluoride membrane (Bio-Rad, Her- cules, CA, USA), used for immunoassay, using ⁇ -actin as the internal reference gene, using automatic image analysis equipment (Li-Cor; Lincoln, NE, USA) to observe and analyze the bands on the film, and the integral of each protein band
  • the optical density is integrated by the normalization method to obtain the corresponding optical density value of the same sample.
  • Statistical analysis is performed using SPSS 19.0 statistical software for statistical analysis.
  • the results of infection of 293T cells by rAAV2-optimized ND4 and rAAV2-ND4 are shown in Figures 6 and 7, respectively.
  • the average expression value of the rAAV2-ND4 group of rabbit optic nerve cells was 0.16, while the average expression value of the rAAV2-optimized ND4 group was 0.48, and the rAAV2-optimized ND4 group was about 3 times that of the rAAV2-ND4 group. There was a significant difference between the two groups (p ⁇ 0.01).
  • the protein level of rAAV2-optimized ND4 group is significantly higher than that of the unoptimized group, which indicates that in terms of translation efficiency, the optimized ND4 encoding nucleotide sequence of the present invention (SEQ ID NO .: 5 85-1464) and the corresponding fusion nucleic acid (SEQ ID NO .: 5 1-2889) translation efficiency is higher.
  • the rabbits in the two groups were examined for slit lamp and intraocular pressure at 1, 3, 7, and 30 days after surgery. All rabbits had no obvious abnormalities, no conjunctival hyperemia, secretions, no endophthalmitis, and no increase in intraocular pressure.
  • the fundus photography of one month after the operation is shown in FIG. 8, where FIG. 8A is the result of fundus photography injected with rAAV2-ND4, and FIG. 8B is the result of fundus photography injected with rAAV2-optimized ND4. It can be seen from the figure that all rabbits have no obvious complications or damage to the retinal blood vessels and optic nerve. It shows that the formal standard intravitreal injection will not cause obvious inflammation or other complications and is safe.
  • the rabbits of the two groups were taken intraocular pressure and fundus photography at 7 days and 30 days after surgery, and the eyeballs were harvested and fixed with eyeball fixation solution. After dehydration, they were embedded in paraffin.
  • the pathological slicer was longitudinally cut along the optic nerve. After further dehydration, it was dyed with hematoxylin and eosin dye solution, and dehydrated and mounted again.
  • FIG. 9A is the HE staining result of rAAV2-ND4 injection
  • FIG. 9B is the HE staining result of rAAV2-optimized ND4 injection. It can be seen from the figure that the retinal ganglion fiber layer of all rabbits is not damaged, and ganglion cells are not reduced. It showed that the formal standard intravitreal injection did not produce retinal toxicity and nerve damage and was safe.
  • the experimental method is the same as that in Embodiment 1-6, and the fusion nucleic acid shown in SEQ ID NO .: 5 is replaced with the fusion nucleic acid shown in SEQ ID NO .: 10 in this embodiment. It was found that compared with the unoptimized ND4 coding sequence, the optimized ND4 coding nucleotide sequence of the present invention (SEQ ID NO: 10 position 267-1646) and the corresponding fusion nucleic acid (SEQ ID NO: 10 1-2271) ND4 transcription efficiency and translation efficiency are significantly improved, the expression level is also significantly higher, can effectively treat Leber hereditary optic neuropathy, and has good safety.

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Abstract

Acide nucléique codant pour une protéine de la sous-unité 4 de la NADH déshydrogénase humaine et application correspondante. La séquence nucléotidique est représentée dans SEQ ID NO : 1. L'invention concerne également un acide nucléique de fusion, comprenant l'acide nucléique codant pour une protéine de la sous-unité 4 de la NADH déshydrogénase humaine. L'invention concerne en outre un vecteur d'expression recombinant contenant l'acide nucléique ou l'acide nucléique de fusion. L'invention concerne par ailleurs un transformant qui introduit l'acide nucléique ou l'acide nucléique de fusion dans un hôte. Le niveau d'expression de l'acide nucléique codant pour une protéine de la sous-unité 4 de la NADH déshydrogénase humaine est plus élevé, ce qui permet d'obtenir plus de protéine de la sous-unité 4 de la NADH déshydrogénase humaine dans les mitochondries, ce qui peut permettre de mieux traiter la neuropathie optique héréditaire de Leber.
PCT/CN2018/118662 2018-06-29 2018-11-30 Séquence codante de protéine nd4 et application correspondante WO2020077756A1 (fr)

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KR1020247001775A KR20240014102A (ko) 2018-06-29 2019-07-01 레버 유전성 시신경병증의 치료를 위한 조성물 및 방법
KR1020217001385A KR102627561B1 (ko) 2018-06-29 2019-07-01 레버 유전성 시신경병증의 치료를 위한 조성물 및 방법
CN202110786772.2A CN113528510A (zh) 2018-06-29 2019-07-01 治疗遗传性视神经病变的组合物和方法
CN202110786630.6A CN113476484A (zh) 2018-06-29 2019-07-01 治疗遗传性视神经病变的组合物和方法
SG11202012044QA SG11202012044QA (en) 2018-06-29 2019-07-01 Compositions and methods for treating leber's hereditary optic neuropathy
MX2020013772A MX2020013772A (es) 2018-06-29 2019-07-01 Composiciones y métodos para el tratamiento de la neuropatía óptica hereditaria de leber.
CA3103740A CA3103740A1 (fr) 2018-06-29 2019-07-01 Compositions et methodes de traitement de la neuropathie optique hereditaire de leber
JP2021521870A JP2021529001A (ja) 2018-06-29 2019-07-01 レーベル遺伝性視神経症を治療するための組成物及び方法
AU2019296451A AU2019296451B2 (en) 2018-06-29 2019-07-01 Compositions and methods for treating leber's hereditary optic neuropathy
CN201980003485.0A CN110876269B (zh) 2018-06-29 2019-07-01 治疗遗传性视神经病变的组合物和方法
PCT/CN2019/094136 WO2020001657A1 (fr) 2018-06-29 2019-07-01 Compositions et méthodes de traitement de la neuropathie optique héréditaire de leber
BR112020026361-3A BR112020026361A2 (pt) 2018-06-29 2019-07-01 Composições e métodos para tratar neuropatia óptica hereditária de leber
EP19826653.8A EP3814492A4 (fr) 2018-06-29 2019-07-01 Compositions et méthodes de traitement de la neuropathie optique héréditaire de leber
EP19853225.1A EP3840785A4 (fr) 2018-08-20 2019-08-20 Compositions et méthodes de traitement de la neuropathie optique héréditaire de leber
AU2019323434A AU2019323434A1 (en) 2018-08-20 2019-08-20 Compositions and methods for treating leber's hereditary optic neuropathy
CA3109432A CA3109432A1 (fr) 2018-08-20 2019-08-20 Compositions et methodes de traitement de la neuropathie optique hereditaire de leber
JP2021509893A JP7403852B2 (ja) 2018-08-20 2019-08-20 レーベル遺伝性視神経症を治療するための組成物及び方法
CN201980054770.5A CN112584874A (zh) 2018-08-20 2019-08-20 用于治疗莱伯氏遗传性视神经病变的组合物和方法
PCT/CN2019/101538 WO2020038352A1 (fr) 2018-08-20 2019-08-20 Compositions et méthodes de traitement de la neuropathie optique héréditaire de leber
KR1020217007727A KR20210068014A (ko) 2018-08-20 2019-08-20 레버 유전성 시신경병증의 치료를 위한 조성물 및 방법
SG11202101032VA SG11202101032VA (en) 2018-08-20 2019-08-20 Compositions and methods for treating leber's hereditary optic neuropathy
US16/836,644 US11034954B2 (en) 2018-06-29 2020-03-31 Compositions and methods for treating leber's hereditary optic neuropathy
US17/181,849 US11352645B2 (en) 2018-08-20 2021-02-22 Compositions and methods for treating Leber's hereditary optic neuropathy
US17/317,295 US20220340895A1 (en) 2018-06-29 2021-05-11 Compositions and methods for treating leber's hereditary optic neuropathy
US17/320,388 US11332741B1 (en) 2018-06-29 2021-05-14 Compositions and methods for treating leber's hereditary optic neuropathy
AU2021204690A AU2021204690A1 (en) 2018-06-29 2021-07-05 Compositions and methods for treating Leber's hereditary optic neuropathy
US17/726,833 US20220259619A1 (en) 2018-08-20 2022-04-22 Compositions and methods for treating leber's hereditary optic neuropathy
JP2023029170A JP2023078173A (ja) 2018-06-29 2023-02-28 レーベル遺伝性視神経症を治療するための組成物及び方法
JP2023205807A JP2024028861A (ja) 2018-08-20 2023-12-06 レーベル遺伝性視神経症を治療するための組成物及び方法
AU2023285773A AU2023285773A1 (en) 2018-06-29 2023-12-20 Compositions and methods for treating Leber's hereditary optic neuropathy

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